Characterization of the complete chloroplast genome sequence of Lycium qingshuiheense (Solanaceae)

Abstract Lycium qingshuiheense is a typical drought and salt-alkali-tolerant plant, which has been added to the new species of Lycium in recent years. Here, we first sequenced the complete chloroplast genome of L. qingshuiheense to investigate its evolutionary relationship within the family Solanaceae. Results suggested that the circular complete chloroplast genome of L. qingshuiheense was 154,945 bp in length, including a large single-copy (LSC) of 85,930 bp, a small single-copy (SSC) of 18,203 bp, and two inverted repeats (IRs) of 25,406 bp. The GC content accounts for 37.90% and annotated 131 genes, including 86 protein-coding genes, eight rRNA genes, and 37 tRNA genes. A neighbor-joining phylogenetic tree revealed that L. qingshuiheense was a sister species to L. ruthenicum. Our study provides a new insight into the systematic evolution of Lycium in the Solanaceae family.


Introduction
Lycium qingshuiheense X. L. Jiang & J. N. Li in 2011, a new species of Lycium in the family Solanaceae, is sparsely distributed in Ningxia and Gansu provinces of northwest China (Li et al. 2011;Cui et al. 2013).The berry of this species appears compressed-globose and darkly red-brown (Li et al. 2011), rich in a variety of vitamins and trace elements (Liu et al. 2016), as well as showing excellent drought tolerance and saline-alkali tolerance.The leaves of L. qingshuiheense are thick and the inner side of the corolla is purple-pink, and calyx with irregularly 2-4 lobed (Figure 1).It has been identified as an important resource of traditional Chinese medicine from the fourth national general investigation of natural resources of Chinese materia medica (Cui et al. 2013).The gene fragments (accD, matk, rps16-trnQ, psbA-trnH, etc.) in plastids are commonly used to mark phylogenetic status and taxonomic attributes of species (Mishra et al. 2015;Van Do et al. 2021).To facilitate its phylogenetic research and contribute to development and utilization, it is urgent to take appropriate measures to protect this rare medicinal resource.In the present study, we determined the complete chloroplast genome of L. qingshuiheense to analyze its phylogenetic position, as well as to provide useful information for further studies.

Materials and methods
Fresh leaves of L. qingshuiheense were sampled from Qingshui River Basin, Zhongning County, Ningxia Hui Autonomous Region, China (37 � 28 0 29.94 N, 105 � 32 0 29.92 E, alt.1150.Wangsuo Liu, email: liuwangsuo@sina.com).Total genomic DNA was extracted according to the modified CTAB method of Stefanova et al. (2013), and stored in the −80 � C refrigerator in the laboratory.The genome sequencing was conducted by Illumina Hiseq 2500 at Biomarker Technologies Corporation (Beijing, China).Filtered the low-quality sequences, and high-quality sequences were assembled by the assembler SPAdes3.11.0 (Nurk et al. 2013).The annotation was performed by Plann (Huang and Cronk 2015).Then, the complete chloroplast genome map was drawn by CPGView program (http:// www.1kmpg.cn/cpgview/)(Liu et al. 2023).Comparison of the junction of LSC, SSC, and IR regions of L. qingshuiheense was performed using R (version 4.2.0) to link to the IRscope website (https://irscope.shinyapps.io/irapp/)(Amiryousefi et al. 2018).The chloroplast genomes of 26 Lycium in Solanaceae and two outgroups were downloaded from the NCBI database.The downloaded chloroplast genome sequence was aligned with L. qingshuiheense using MAFFT-7.037(Katoh and Standley 2013).A neighbor-joining phylogenetic tree was constructed by MEGA-X (Kumar et al. 2018) based on 28 species.The sequence of L. qingshuiheense complete chloroplast genome has been submitted to GenBank (accession number OR551484).

Discussion
Previous studies have reported that the complete chloroplast genome of L. barbarum, L. chinense, and L. ruthenicum were 155,656, 155,745, and 154,869 bp in length, respectively, each containing 133 genes (Cui et al. 2019).In our study, the length of L. qingshuiheense was 154,945 bp, which was close to that of L. ruthenicum, but contains 131 genes.Three regions with high variation, atpH-atpI, accD-ycf4, and ndhF-trnL, were found in L. barbarum, L. chinense, and L. ruthenicum, and these regions can be used to mark and identify systematic variation among these species (Cui et al. 2019), future studies of our study will focus on the analysis and comparison of high variable regions between L. qingshuiheense and other Lycium to explore their phylogenetic differences.Boundary expansion and contraction are considered to be the key reasons for the occurrence of high-variation regions (Konhar et al. 2019).The IRa/SSC and Irb/SSC boundaries are generally positioned in the coding region of ycf1 and rps19 genes, which is easy to cause the duplication of these genes, resulting in pseudogenes with different sizes of these boundaries, it is similar to the results of Cui et al. (2019).In general, the contractive and expansive boundaries of L. qingshuiheense were similar to those of L. ruthenicum, and the source of their migration was likely closer.It indicates the similarity and uniqueness of chloroplast genomes between L. qingshuiheense and its closest relatives in phylogenetic evolution.

Conclusions
In this study, the chloroplast genome of L. qingshuiheense was first sequenced, and the annotated circular structure map was obtained.The data of L. qingshuiheense are of great reference value for the evaluation of genetic variation, phylogeny, classification recognition, and DNA markers of this species.In addition, this study is also a supplement and improvement of the evolutionary data of Lycium species, which lays a foundation for future research.

Figure 2 .
Figure 2. Gene map of L. qingshuiheense chloroplast genome.Genes shown inside the circle indicate that the direction of transcription is clockwise, while those shown outside are counterclockwise.Different groups of functional genes are indicated in different colors.The GC content is shown in the dashed area in the inner circle.